Droplet Actuator with Improved Top Substrate

ABSTRACT

The invention provides a droplet actuator comprising a base substrate and a top substrate separated to form a gap, wherein: (a) the base substrate comprises electrodes configured for conducting droplet operations in the gap; and (b) the top substrate comprises a first portion coupled to second portion, where the second portion comprises one or more openings establishing a fluid path extending from an exterior of the droplet actuator and into the gap. The invention also provides related methods of manufacturing the droplet actuator, methods of using the droplet actuator, and methods of loading the droplet actuator.

RELATED PATENT APPLICATIONS

This application claims priority to U.S. Patent Application No.60/969,757, filed on Sep. 4, 2007, entitled “Improved droplet actuatorloading”; and U.S. Patent Application No. 60/980,785, filed on Oct. 18,2007, entitled “Droplet actuator with improved top plate”; the entiredisclosures of which is incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with government support under NNJ06JD53C awardedby the National Aeronautics and Space Administration of the UnitedStates. The United States Government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates to droplet actuation devices, and in particular tospecialized structures for conducting droplet operations.

BACKGROUND

Droplet actuators are used to conduct a wide variety of dropletoperations. A droplet actuator typically includes two substratesseparated by a gap. The substrates are associated with electrodes forconducting droplet operations. The gap includes a filler fluid that isimmiscible with the fluid that is to be manipulated on the dropletactuator. The formation and movement of droplets in the gap iscontrolled by electrodes for conducting a variety of droplet operations,such as droplet transport and droplet dispensing. At least one of thesurfaces is typically made from a transparent material, such as a glasstop substrate. Among other things, when glass is used, adding featuresto the glass, such as openings for loading fluid into the gap, can becomplex and expensive. There is a need for alternative droplet actuatorstructures that are easier and less expensive to manufacture whileproviding the same or better functionality as glass top substrates.

SUMMARY OF THE INVENTION

The invention provides a modified droplet actuator. The droplet actuatorgenerally includes a base substrate and a top substrate separated toform a gap. One or both substrates, but typically the base substrate,includes electrodes configured for conducting droplet operations in thegap. The top substrate may include a first portion coupled to secondportion, where the second portion includes one or more openingsestablishing a fluid path extending from an exterior of the dropletactuator and into the gap.

The first portion may include a more uniformly planar surface exposed tothe gap than the second portion. In some embodiments, the first portionis more transparent than the second portion, or the first portion istransparent and the second portion is not. In one embodiment the firstportion is substantially transparent, and the second portion issubstantially opaque. In another embodiment, the first portion harderthan the second portion. In still another embodiment, the first portionis more thermally stable than the second portion. In yet anotherembodiment, the first portion is more resistant to damage caused bytemperature fluctuation than the second portion.

The invention also provides a droplet actuator including a basesubstrate and a top substrate separated to form a gap, wherein the basesubstrate includes electrodes configured for conducting dropletoperations in the gap; and the top substrate includes a glass portioncoupled to a non-glass portion, where the non-glass portion includes oneor more openings establishing a fluid path extending from an exterior ofthe droplet actuator and into the gap. The non-glass portion may, insome embodiments, include or be manufactured from a plastic or resinportion. In some cases, the non-glass portion includes a frame intowhich the glass portion is inserted.

The fluid path may be arranged to flow fluid into an actual or virtualreservoir associated with one or more reservoir electrodes associatedwith the base substrate. The fluid path may be arranged to flow fluidinto proximity with one or more of the electrodes.

In some embodiments, the glass portion does not include openingstherein. In some embodiments, the non-glass portion overlaps the glassportion, and an aperture is provided in the non-glass portion forproviding a sensing path from the gap, through the glass portion,through the aperture to an exterior of the droplet actuator. A fittingmay be provided in association with the aperture for fitting a sensoronto the droplet actuator.

In some embodiments, a handle is provided, extending from the glassportion and arranged to facilitate user handling of the dropletactuator. In other embodiments, the non-glass portion further includes ahinged cover arranged to seal the openings when the hinged cover is in aclosed position. The cover may include one or more dried reagentsassociated therewith, such that when fluid is present in one or more ofthe openings, and the cover is closed, the dried reagents contact thefluid and are combined therewith to form fluid reagents.

In another embodiment, the non-glass portion overlaps the glass portion;and one or more of the openings extends through the non-glass portion,through the glass portion, and into the gap. In some embodiments, theopening extending through the non-glass portion is configured as a fluidreservoir.

The invention also provides a droplet actuator including a basesubstrate and a top substrate separated to form a gap, wherein the (a)base substrate includes electrodes configured for conducting dropletoperations in the gap; and an opening forming a fluid path from anexterior of the droplet actuator into the gap; and (b) the top includesa top substrate electrode arranged opposite the opening such that fluidflowing into the gap through the opening flows into proximity with thetop substrate electrode.

The invention also includes methods of loading a fluid onto a dropletactuator. The methods generally include providing a droplet actuator ofthe invention and loading a fluid through the opening and into the gap.

The invention also includes methods of assembling a droplet actuator ofthe invention. The methods generally coupling the glass portion to thenon-glass portion of the top substrate, and assembling the top substratewith the bottom substrate to form a gap therebetween suitable forconducting droplet operations.

Finally, the invention includes methods of conducting a dropletoperation. The methods generally include providing a droplet actuator ofthe invention; loading a liquid onto the droplet actuator into proximitywith one or more electrodes; and using the one or more electrodes toconduct the droplet operation.

Other aspects of the invention will be apparent from the ensuingdetailed description of the invention.

DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may, forexample, be aqueous or non-aqueous or may be mixtures or emulsionsincluding aqueous and non-aqueous components. Droplets may take a widevariety of shapes; nonlimiting examples include generally disc shaped,slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, ovoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplets, see U.S. Pat. No. 6,911,132, entitled “Apparatusfor Manipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat.No. 6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No.6,565,727, entitled “Actuators for Microfluidics Without Moving Parts,”issued on Jan. 24, 2000, both to Shenderov et al.; Pollack et al.,International Patent Application No. PCT/US2006/047486, entitled“Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures ofwhich are incorporated herein by reference. Methods of the invention maybe executed using droplet actuator systems, e.g., as described inInternational Patent Application No. PCT/US2007/009379, entitled“Droplet manipulation systems,” filed on May 9, 2007. In variousembodiments, the manipulation of droplets by a droplet actuator may beelectrode mediated, e.g., electrowetting mediated or dielectrophoresismediated.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; condensing a dropletfrom a vapor; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet actuator; other droplet operations describedherein; and/or any combination of the foregoing. The terms “merge,”“merging,” “combine,” “combining” and the like are used to describe thecreation of one droplet from two or more droplets. It should beunderstood that when such a term is used in reference to two or moredroplets, any combination of droplet operations sufficient to result inthe combination of the two or more droplets into one droplet may beused. For example, “merging droplet A with droplet B,” can be achievedby transporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading. In various embodiments, thedroplet operations may be electrode mediated, e.g., electrowettingmediated or dielectrophoresis mediated.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. The filler fluid may, forexample, be a low-viscosity oil, such as silicone oil. Other examples offiller fluids are provided in International Patent Application No.PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec.11, 2006; and in International Patent Application No. PCT/US2008/072604,entitled “Use of additives for enhancing droplet actuation,” filed onAug. 8, 2008.

The terms “top” and “bottom,” when used, e.g., to refer to the top andbottom substrates of the droplet actuator, are used for convenienceonly; the droplet actuator is generally functional regardless of itsposition in space.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet actuator forconvenience only, since the droplet actuator is functional regardless ofits position in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

DESCRIPTION

The invention provides a droplet actuator with improved features forloading fluid into the gap. In certain embodiments, the droplet actuatorincludes a top substrate that combines glass with one or more othermaterials that are easier to manufacture. Examples of such materialsinclude resins and plastics. One such embodiment includes a topsubstrate including a glass substrate portion and a plastic portion. Theglass substrate portion covers the droplet operations area of thedroplet actuator, providing a flat, smooth surface for facilitatingeffective droplet operations. The plastic portion has one or moreopenings that provide a fluid path from an exterior locus into the gapof the droplet actuator. The fluid path facilitates loading of fluidinto the gap of the droplet actuator.

An alternative embodiment of the invention provides a droplet actuatorwith one or more openings in the bottom substrate or substrate. Variousembodiments of the invention may reduce or eliminate the need to formopenings in the glass portion of a droplet actuator, avoiding a complexand costly manufacturing step. Still other embodiments avoid the use ofglass altogether.

It should also be noted that in various embodiments, the non-glassportion may include multiple kinds of plastics rather than aglass/non-glass construction. For example, in the variousglass/non-glass embodiments, one plastic may be substituted for theglass component and a second plastic may be used for the non-glasscomponents. This approach may be employed to, among other things, takeadvantage of different optical properties (e.g., opaque forreservoirs/clear over electrodes or over detection zones) mechanicalproperties (flat, hard, planar, precise over electrodes/cheap, easy tomold or machine for fluid passages into reservoirs) or thermalproperties (high T over electrodes for film deposition or PCR/cheaperlow T for wells), surface properties and the like. In yet anotheralternative embodiment, the glass portion may be replaced with or coatedwith a metal foil and a non-glass material may be provided in regionswhere fluid passages into the droplet actuator are desired, for ease ofmanufacture.

7.1 Loading Mechanisms Using a Modified Top Substrate

FIGS. 1A and 1B illustrate a top view and cross-sectional view,respectively, of an embodiment of a droplet actuator 100. FIG. 1B is across-sectional view that is taken along line A-A of FIG. 1A.

Droplet actuator 100 includes a top substrate 110 that combines a glassportion with a second material, such as resin or plastic. In oneembodiment, the top substrate 110 is formed of a glass substrate 114,the perimeter of which is partially or completely surrounded by anon-glass (e.g., plastic or resin) frame 118. The frame 118 includes oneor more openings 122 forming a fluid path from an exterior of thedroplet actuator 100 into the gap 132. In some embodiments, one or moreof the openings 122 may provide a fluid path extending from the exteriorof the droplet actuator 100 into an actual or virtual reservoirassociated with one or more reservoir electrodes 134. In otherembodiments, one or more of the openings 122 may provide a fluid paththat is not aligned with or associated with any electrode or with anyspecialized electrode, such as a reservoir electrode.

Additionally, droplet actuator 100 includes a bottom substrate 126. Thebottom substrate 126 includes an associated arrangement of electrodes130 for performing droplet operations. Electrodes 130 may, for example,be covered with a hydrophobic insulator to permit manipulation of theliquid by electrowetting. The bottom substrate may also include one ormore reservoir electrodes 134 for use in dispensing fluid from thereservoir. Bottom substrate 126 may, for example, be made using printedcircuit board (PCB) technology or semiconductor manufacturingtechnology. Top substrate 110 and bottom substrate 126 are separatedfrom one another to form a gap for conducting droplet operations.

The area of glass substrate 114 of top substrate 110 may be selected tocover the active droplet manipulation area of droplet actuator 100. Inone example, the area of glass substrate 114 may substantially cover thearrangement of electrodes 130. The locations of openings 122 of frame118 may correspond with locations of the one or more reservoirelectrodes 134. In one embodiment, one or more reservoir electrodes ispositioned at the periphery of glass substrate 114 for drawing aquantity of fluid 138 through the openings 122 into droplet actuator100, e.g., as shown in FIG. 1B. In another embodiment, one or morereservoir electrodes is positioned at the periphery of glass substrate114 and overlaps with glass substrate 114 for drawing a quantity offluid 138 through the openings 122 into droplet actuator 100. Frame 118may be bonded to the periphery edges of glass substrate 114 usingadhesives or may be manufactured to permit glass substrate to be snuglyfitted into place.

Glass substrate 114 may be transparent. Ideally, glass substrate 114 isas thin as is practical for providing optimal droplet detectioncapabilities. Frame 118 may, in some embodiments, be opaque and may besubstantially the same thickness or thicker than glass substrate 114. Athick frame 118 may facilitate including fluid reservoirs or wellsassociated with openings 122 to contain a volume of fluid. Becauseopenings 122 are formed within frame 118, glass substrate 114 may bemanufactured without the need for forming openings therein. As a result,the added cost and complexity of forming openings in a glass topsubstrate may be reduced, preferably entirely avoided. By contrast, theprocess for forming openings, such as fluid reservoirs 122, in a plasticstructure, such as frame 118, may be simple and inexpensive. In oneembodiment, the total amount of glass required in the device isminimized by only using glass where the flatness, and optical qualitiesare required.

FIG. 2A illustrates a side view of a droplet actuator 200 havinggenerally the same characteristics as droplet actuator 100 shown inFIG. 1. Additionally, in droplet actuator 200, the frame 122 partiallyoverlies the glass substrate 214 forming an overlapping substrate 218and leaving one or more openings 238 sized to permit detection ofdroplet characteristics through the glass substrate 214. The locationsof the one or more apertures 238 may correspond to detection areas(e.g., certain of the electrodes 230) within droplet actuator 200 wheredetection is to take place.

FIG. 2B illustrates another side view of a droplet actuator 200 that isdescribed in FIG. 2A. However, FIG. 2B shows the addition of analignment structure 242 that is coupled to substrate 218 of dropletactuator 200 at aperture 238. Alignment structure 242 may be formed of,for example, molded plastic. In one example, the purpose of alignmentstructure 242 may be to align aperture 238 of droplet actuator 200 witha corresponding alignment structure 246 associated with an externaloptical detector 246. The shape of alignment structure 240 may, forexample, selected to provide for easy alignment with a cavity ofexternal alignment structure 246.

FIG. 3 illustrates a top view of a top substrate 310 that issubstantially the same as top substrate 110 of droplet actuator 100 ofFIGS. 1A and 1B, except for the addition of a handle 314, which may insome embodiments be molded with the non-glass (e.g., plastic or resin)portions of top substrate 110. Handle 314 may be formed to extend fromthe main body (i.e., the active droplet operations area) of topsubstrate 310, in order to facilitate handling of the droplet actuator.

FIG. 4 illustrates a side view of a droplet actuator 400 that issubstantially the same as droplet actuator 100 of FIGS. 1A and 1B and/ordroplet actuator 200 of FIGS. 2A and 2B, except for the addition of acover 410. Cover 410 may be attached to frame 118 via a hinge 414, whichprovides an easy opening and closing mechanism. Optionally, cover 410may include one or more dried reagents 418 that correspond with openings122 so that when fluid is included in the reservoirs and cover 410 isclosed, the dried reagents are reconstituted in the fluid. Cover 410 maybe formed to seal fluid reservoirs 122 when closed. In some embodiments,cover 410 may be molded together with frame 118 as a unitary structure.

7.2 Top Substrate Assemblies

FIGS. 5A, 5B, and 5C illustrate cross-sectional views of dropletactuators that include various embodiments of a loading mechanism thatemploys a top substrate made from glass and non-glass components.

In one embodiment, FIG. 5A illustrates cross-sectional view of a dropletactuator 500 that includes a top substrate 510 that is formed of a glasssubstrate 514 and a frame 518. Additionally, droplet actuator 500includes a bottom substrate 522 that has an associated arrangement ofelectrodes. Top substrate 510 and bottom substrate 522 are arranged toform a gap for conducting droplet operations. Glass substrate 514 may besubstantially the same as glass substrate 114 of droplet actuator 100 ofFIGS. 1A and 1B. Similar to frame 118 of droplet actuator 100, frame 518may include one or more openings (not shown) and a clearance region thatcorresponds to the active droplet operations area of droplet actuator500 for fitting a glass substrate, such as glass substrate 514, therein.However, differing from frame 118 of droplet actuator 100, the crosssection of frame 518 provides an L-shaped structure, which provides aside wall for surrounding the active droplet operations area of dropletactuator 500 and which also provides a top surface to which glasssubstrate 514 may abut. Additionally, an arrangement of spacers 526 areprovided between glass substrate 514 and bottom substrate 522, in orderto support glass substrate 514 against frame 518. When assembled, glasssubstrate 514, frame 518, and spacers 526 define the gap of dropletactuator 500. The height of the walls of frame 518 and spacers 526correspond to a desired gap height.

In another embodiment, FIG. 5B illustrates a cross-sectional view of adroplet actuator 530. droplet actuator 530 is substantially the same asdroplet actuator 500 of FIG. 5A, except that top substrate 510 isreplaced by top substrate 534. Top substrate 534 includes glasssubstrate 514 of FIG. 5A and a frame 538. Integrated spacers 542, whichreplace spacers 526 of FIG. 5A, are provided as part of the structure offrame 538. Additionally, the integration of built-in spacers 542 withinframe 538 forms a groove 546 into which glass substrate 514 may beinstalled. Again, the height of built-in spacers 542 corresponds to adesired gap height.

In yet another embodiment, FIG. 5C illustrates a cross-sectional view ofa droplet actuator 550. droplet actuator 550 is substantially the sameas droplet actuator 530 of FIG. 5B, except that top substrate 534 isreplaced by top substrate 544. Top substrate 544 includes glasssubstrate 514 of FIG. 5A and a substrate 548. Substrate 548 may formedwith frame 538, including integrated spacers 542 and groove 546.However, substrate 548 differs from frame 538 in that it does notinclude the opening. Instead, when installed in groove 546, glasssubstrate 514 is fully covered by substrate 548. Again, the height ofbuilt-in spacers 542 corresponds to a desired gap height.

Referring again to FIGS. 5A, 5B, and 5C, the assemblies may includeother features, such as tooling openings, in both the glass andnon-glass portions of the top substrate. In one example, the toolingopenings may accommodate nuts and bolts for holding the assembliestogether.

FIG. 6 illustrates a cross-sectional view of a droplet actuator 600 thatincludes another non-limiting example of a loading mechanism that uses acombination glass and non-glass (e.g., plastic and/or resin) topsubstrate. Droplet actuator 600 includes a top substrate 610 that isformed of a glass substrate 614 that may be coupled to a non-glass frame618. Additionally, droplet actuator 600 includes a bottom substrate 622that includes an associated arrangement of electrodes. Top substrate 610and bottom substrate 622 are arranged to provide a gap for conductingdroplet operations.

Glass substrate 614 further includes one or more openings 626 thatcorrespond to one or more fluid reservoirs 632 within frame 618, asshown in FIG. 6, for the purpose of loading droplet actuator 600. Thisembodiment includes openings that are formed in both glass substrate 614and non-glass frame 618, which differs from the embodiments of FIGS. 1Athrough 5C.

In this embodiment, because of the structural support that is providedby non-glass frame 618, the thickness of glass substrate 614 may beminimized, which allows the glass drilling process to be simplified. Inorder to facilitate easy loading or to provide reservoirs of largerfluid capacity, fluid reservoirs 632 of frame 618 may be larger thanopenings 626 of glass substrate 614. Additionally, the walls of fluidreservoirs 632 of frame 618 may have any of a variety of configurations,such as vertical walls or tapered (e.g., to form a conical shape) from alarge opening to the smaller openings 626 of glass substrate 614.Forming such shapes in glass would be difficult, but is readily achievedusing materials such as plastic or resins. Additionally, frame 618 maybe provided having any useful thickness, thereby providing any usefulfluid capacity via reservoirs 632.

In yet another embodiment, any of the foregoing embodiments may replacethe glass portion with a molded material, such as a plastic or resin.Further, any of the foregoing embodiments may be made as a singleplastic or resin component, rather than as glass/non-glass components.

In yet other embodiments, the top substrate may include one or moreoptical elements formed therein. For example, the optical element mayinclude a lens and/or a diffraction gradient. The optical element may beconfigured to redirect, or otherwise modify, light to or from a droplet,fluid or surface of a droplet actuator. The optical element may be amodification in a surface of the top substrate or a coating adhered toor layered on a surface of the top substrate.

In one embodiment, the invention provides a top or bottom substrate thatincludes optical surface patterning. The optical surface patterning maybe provided in a glass or non-glass portion of the top or bottomsubstrate. The top or bottom substrate may itself be glass or acombination of glass/non-glass. The optical surface patterning may, forexample, introduce a diffractive optical element to the modifiedsubstrate. In one embodiment, the diffractive optical element introducessurface features on the same order of magnitude as the wavelength oflight (micrometers or smaller) used for detection purposes. The opticalsurface patterning may be selected so that diffractive effects dominaterefractive effects. In this manner, the microstructure of the opticalsurface patterning breaks up the light wave in a manner which producesinterference patterns. The interference patterns can be evaluated todetermine the shape of the output waveform.

7.3 Loading Mechanism in a Bottom Substrate

FIG. 7 illustrates cross-sectional view of a droplet actuator 700 thatincludes a non-limiting example of a loading mechanism in the bottomsubstrate thereof Droplet actuator 700 includes a first substrate 710that includes at least one reservoir electrode 714. Additionally,droplet actuator 700 includes a second substrate 718 that is formed of asubstrate 722 that has an associated arrangement of electrodes 726,e.g., electrowetting electrodes, for performing droplet operations. Thesubstrate 722 may, for example, be a PCB substrate. First substrate 710and second substrate 718 are arranged to form a gap for conductingdroplet operations.

In this example, at least one opening 730 is provided in the secondsubstrate, e.g., as shown in FIG. 7. Opening 730 may serve as an inletfor loading the reservoir of droplet actuator 700. When droplet actuator700 is initally loaded with liquid, the liquid body may not reach theextent of electrodes 726 (and therefoe be manipulated by theseelectrodes) owing to the fact that the electrodes and inlet are on thesame side of substrate 722 and that a certain amount of separation mustbe maintained between the edge of opening 730 and the edge of electrode726. This situation can be improved through the use of a reservoirelectrode 714 located on the opposite substrate 710 and positioned tosubstantially align with opening 730. The geometry of reservoirelectrode 714 may overlap slightly with the electrodes 726 that are oneither side of opening 730 of second substrate 718. Additionally,reservoir electrode 714 is electrically isolated from the ground (notshown).

In operation, droplet actuator 700 may be held in an invertedorientation, such as shown in FIG. 7, and a quantity of fluid 734 may bedrawn into droplet actuator 700 via opening 730 within substrate 722 byactivating reservoir electrode 714 to bring the liquid into theproximity of electrode 726. Once loaded, reservoir electrode 714 isdeactivated and the fine control for performing droplet operations isperformed via electrodes 726 of substrate 718. The PCB embodiment ofFIG. 7 has the advantage of a low cost, standard process for formingopenings and also allows for high precision when forming openings.

7.4 Combined Cartridge/Sample Collection Device

The modified substrates of the invention may also be used to providesample collection functionality to a droplet actuator cartridge. Forexample, the top or bottom substrate may be associated with a syringefor sampling a liquid, such as blood or water. The syringe collectionchamber may itself serve as liquid reservoir on the top or bottomsubstrate of the droplet actuator. In this embodiment, the top or bottomsubstrate includes or is associated with a fluid path from the gapbetween the substrate into the syringe collection chamber. Liquid fromthe collection chamber flows through the fluid path into proximity toone or more droplet operations electrodes, where it can be subjected toone or more droplet operations. Other embodiments may include simplesample collection tubes or catheters for introducing liquid from anexterior source into a droplet actuator for analysis.

In another embodiment, the droplet actuator may be configured to serveas a combination forensic sample collection tube and analysis cartridge.Microfluidic analysis can be performed either in the field, e.g., at thepoint of sample collection, or in a central lab. This configurationprovides a quick test result while maintaining the bulk of the sample inpristine condition for further forensic testing. Follow-up testing forevidentiary purposes can then be performed later on the same sampleusing conventional (i.e., legally-accepted) techniques. In a relatedembodiment, the droplet actuator includes a break-away sample storagecomponent so that the sample can be preserved in a more compact form.

7.5 Fluids

For examples of fluids that may be subjected to the loading operationsand droplet operations using the modified droplet actuators of theinvention, see the patents listed in International Patent ApplicationNo. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed onDec. 11, 2006. In some embodiments, the fluid includes a biologicalsample, such as whole blood, lymphatic fluid, serum, plasma, sweat,tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminalfluid, vaginal excretion, serous fluid, synovial fluid, pericardialfluid, peritoneal fluid, pleural fluid, transudates, exudates, cysticfluid, bile, urine, gastric fluid, intestinal fluid, fecal samples,fluidized tissues, fluidized organisms, biological swabs and biologicalwashes. In some embodiment, the fluid includes a reagent, such as water,deionized water, saline solutions, acidic solutions, basic solutions,detergent solutions and/or buffers. In other embodiments, the fluidincludes a reagent, such as a reagent for a biochemical protocol, suchas a nucleic acid amplification protocol, an affinity-based assayprotocol, a sequencing protocol, and/or a protocol for analyses ofbiological fluids.

7.6 Method of making and Loading a Droplet Actuator of the Invention

A method of making a droplet actuator that includes a combinationglass/non-glass top substrate includes, but is not limited to, the stepsof (1) forming a bottom substrate from, for example, a PCB that includestransport electrodes and also one or more reservoir electrodes at itsperiphery; (2) forming a glass substrate the corresponds to the activeelectrowetting area of the bottom substrate of the droplet actuator; (3)forming a non-glass (e.g., plastic or resin) frame or substrate, towhich the glass substrate may be coupled, and wherein the frame orsubstrate includes one or more fluid paths for introducing fluid intothe gap; (4) assembling the bottom substrate and top substrate one toanother to form the gap. Loading may involve providing a quantity offluid through the fluid path into the gap. Where the fluid being loadedis a sample or reagent, the fluid may be loaded into proximity with anelectrode so that droplet operations may be conducted using the fluid.

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. Thisspecification is divided into sections for the convenience of the readeronly. Headings should not be construed as limiting of the scope of theinvention. The definitions are intended as a part of the description ofthe invention. It will be understood that various details of the presentinvention may be changed without departing from the scope of the presentinvention. Furthermore, the foregoing description is for the purpose ofillustration only, and not for the purpose of limitation, as the presentinvention is defined by the claims as set forth hereinafter.

1. A droplet actuator comprising a base substrate and a top substrateseparated to form a gap, wherein: (a) the base substrate compriseselectrodes configured for conducting droplet operations in the gap; and(b) the top substrate comprises a first portion coupled to secondportion, where the second portion comprises one or more openingsestablishing a fluid path extending from an exterior of the dropletactuator and into the gap.
 2. The droplet actuator of claim 1 whereinthe first portion comprises a more uniformly planar surface exposed tothe gap than the second portion.
 3. The droplet actuator of claim 1wherein the first portion is more transparent than the second portion.4. The droplet actuator of claim 1 wherein the first portion issubstantially transparent, and the second portion is substantiallyopaque.
 5. The droplet actuator of claim 1 wherein the first portionharder than the second portion.
 6. The droplet actuator of claim 1wherein the first portion is more thermally stable than the secondportion.
 7. The droplet actuator of claim 1 wherein the first portion ismore resistant to damage caused by temperature fluctuation than thesecond portion.
 8. A droplet actuator comprising a base substrate and atop substrate separated to form a gap, wherein: (a) the base substratecomprises electrodes configured for conducting droplet operations in thegap; and (b) the top substrate comprises a glass portion coupled to anon-glass portion, where the non-glass portion comprises one or moreopenings establishing a fluid path extending from an exterior of thedroplet actuator and into the gap.
 9. The droplet actuator of claim 8wherein the non-glass portion comprises a plastic or resin portion. 10.The droplet actuator of claim 8 wherein the non-glass portion comprisesa frame into which the glass portion is inserted.
 11. The dropletactuator of claim 8 wherein the fluid path is arranged to flow fluidinto an actual or virtual reservoir associated with one or morereservoir electrodes associated with the base substrate.
 12. The dropletactuator of claim 8 wherein the fluid path is arranged to flow fluidinto proximity with one or more of the electrodes.
 13. The dropletactuator of claim 8 wherein the glass portion does not include openingstherein.
 14. The droplet actuator of claim 8 wherein: (a) the non-glassportion overlaps the glass portion, and (b) an aperture is provided inthe non-glass portion for providing a sensing path from the gap, throughthe glass portion, through the aperture to an exterior of the dropletactuator.
 15. The droplet actuator of claim 14 further comprising afitting provided in association with the aperture for fitting a sensoronto the droplet actuator.
 16. The droplet actuator of claim 14 furthercomprising a handle extending from the glass portion and arranged tofacilitate user handling of the droplet actuator.
 17. The dropletactuator of claim 8 wherein the non-glass portion further comprises ahinged cover arranged to seal the openings when the hinged cover is in aclosed position.
 18. The droplet actuator of claim 17 wherein the hingedcover comprises one or more dried reagents associated therewith, suchthat when fluid is present in one or more of the openings, and the coveris closed, the dried reagents contact the fluid and are combinedtherewith to form fluid reagents.
 19. The droplet actuator of claim 8wherein: (a) the non-glass portion overlaps the glass portion; and (b)one or more of the openings extends through the non-glass portion,through the glass portion, and into the gap.
 20. The droplet actuator ofclaim 19 wherein the opening extending through the non-glass portion isconfigured as a fluid reservoir.
 21. A droplet actuator comprising abase substrate and a top substrate separated to form a gap, wherein: (a)the base substrate comprises: (i) electrodes configured for conductingdroplet operations in the gap; and (ii) an opening forming a fluid pathfrom an exterior of the droplet actuator into the gap; and (b) the topcomprises a top substrate electrode arranged opposite the opening of(a)(ii) such that fluid flowing into the gap through the opening of(a)(ii) flows into proximity with the top substrate electrode.
 22. Amethod of loading a fluid onto a droplet actuator, the method comprisingproviding a droplet actuator of claim 1 and loading a fluid through theopening and into the gap.
 23. A method of assembling the dropletactuator of claim 1, the method comprising: (a) coupling the glassportion to the non-glass portion; and (b) assembling the top substratewith the bottom substrate to form a gap therebetween suitable forconducting droplet operations.
 24. A method of conducting a dropletoperation, the method comprising: (a) providing a droplet actuator ofclaim 1; (b) loading a liquid onto the droplet actuator into proximitywith one or more electrodes; (c) using the one or more electrodes toconduct the droplet operation.